In molecular biotechnology and bioengineering, the production of recombinant protein is achieved by cloning the target gene into an expression vector, and introducing the recombinant vector into corresponding host such as bacterium, yeast, plant or animal cells, where the target gene is expressed. Bacterial host E. coli is often the first choice for the expression of many recombinant proteins, because it is easy, fast and inexpensive to cultivate, and its vector systems have been well developed. To reach a high level of expression in E. coli, the foreign gene is usually under the control of a regulatory promoter, which plays important roles in reducing the adverse effects of recombinant protein on host cells, decreasing the degradation of target protein by cellular protease of the host cells, and increasing the production of active recombinant protein. Using promoters of different sources, many E. coli expression vector systems have been developed in the last 20 years, and the best known vectors are those containing lac promoter and its hybrids, the bacteriophage λ pL promoter and T7 promoter, which are respectively identified as the lac/tac/trc system, the pL system and the T7 system (Sambrook, J, and D W Russell. 2001. Molecular Cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
The lac/tac/trc system. In this expression system, vectors carry the lac promoter, or its hybrid tac or trc promoter. Under the control of one of these promoters, the transcription initiation of target gene is repressed by the repressor of lac opron in the absence of lactose or its analogues such as isopropyl-β-D-thiogalactopyranoside (IPTG). In both experimental and commercial production settings, the expression of target genes is induced by adding IPTG or lactose as the inducing agent into the culture of E. coli harboring vectors of the lac/tac/trc system, wherein the inducing agent releases the repressor and allows transcription to initiate. However, the high cost and toxicity of IPTG limites its wide use for the production of proteins for medical and many industrial applications. Lactose is cheaper than IPTG as inducing agent, but is not as effective as the latter because it can be metabolized.
The pL system. In bacteriophage λ, the early transcription promoters pL and pR are regulated by a repressor encoded by the cI gene. The pL has been used in expression vectors to control the expression of target genes via the gene product of cIts857, which is a temperature sensitive mutant of cI. In cells harboring these vectors, the repressor binds to pL and represses the transcription of target gene at low temperatures but not at elevated temperatures, and thus gene expression is induced by raising the temperature of a culture (Elvin C M, P R Thompson, M E Argall et al. 1990. Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene, 87: 123-126). However, because an effective induction requires rapidly raising the temperature from about 30° C. to 40° C., there is difficulty in the application of the pL system for large-scale cultures in industrial settings (Glazer, A N, and H Nikaido. 1995. Microbial Biotechnology. WH Freeman and Company, New York).
The T7 system. In this system, the bacteriophage T7 promoter is used in vectors to control the expression of target gene, and the transcription is specifically performed by T7 RNA polymerase. The gene encoding T7 RNA polymerase has been integrated in the chromosome of host cells under the control of lac or pL promoter, and its expression is induced by IPTG or temperature shift. The bacteriophage T7 promoter is the strongest among all the promoters used in E. coli expression systems, but growth inhibition or inclusion body formation sometimes are associated with high expression levels [Russell D. 1999. Gene expression systems based on bacteriophage T7 RNA polymerase. In Gene Expression Systems (Fernandez, J M, and J P Hoeffler, eds.), pp 9-44. Academic Press, London]. Meanwhile, the T7 system faces the same problems as other systems in inducing agents or raising temperatures.
The heat shock system of E. coli. When E. coli is subjected to a quick rise of temperature, an alternative sigma factor (σ32, encoded by the rpoH gene) recognizes the so-called heat-shock promoters of a group of heat-shock protein-encoding genes, resulting in the expression of heat-shock proteins. The DNA sequences of heat-shock promoters have been known, and their consensus sequences are different from that of the general promoters recognized by the σ70 (Miller, J H. 1992. A Short Course in Bacterial Genetics, Handbook. Cold Spring Harbor Laboratory Press, New York) (Turner, P C T, A G McLennan, A D Bates, and M R H White. 1997. Instant Notes in Molecular Biology, BIOS Scientific Publishers, UK). The differences of the two consensus sequences are shown below.
General promoters:(SEQ ID NO: 11)-----------TTGACA-16~18 bp-TATAAT Heat-shock promoters:(SEQ ID NO: 12)--C-C-CTTGAA-13~15 bp-CCCUCAT-T
Although the heat-shock system in E. coli has been well understood for its physiological functions and regulatory mechanisms, prior to the present invention, heat-shock promoters have never been used effectively as promoters to regulate the expression of foreign genes in plasmid vectors. This may be due to the fact that the heat-shock reaction lasts (i.e. the heat-shock system shuts down, and the cell is back to its normal state within 20 min) only 20 minutes after E. coli is subjected to an increase in temperature. Commercial application of this system may have also been discouraged by the apparent difficulty in quickly raising the temperature of large volume of culture medium, as in the case of using pL system.